reaction mass of 3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 1-(2,6,6-trimethylcyclohex-2-en-1-yl)pent-1-en-3-one

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jan 2021 - 02 Jun 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals: 10 weeks; according to ECHA Compliance Check Decision CCH-D-2114359360-53-01/F
- Basis for dose level selection: Based on a range finding study
- Inclusion of extension of Cohort 1B: yes; according to ECHA Compliance Check Decision CCH-D-2114359360-53-01/F
- Termination time for F2: terminal sacrifice of the F2 weanlings.
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: according to ECHA Compliance Check Decision CCH-D-2114359360-53-01/F
- Exclusion of developmental immunotoxicity Cohort 3: according to ECHA Compliance Check Decision CCH-D-2114359360-53-01/F
- Route of administration: oral (gavage): according to ECHA Compliance Check Decision CCH-D-2114359360-53-01/F
- Choice of species, strain: The rat is the preferred animal species for reproduction studies according to test guidelines. This strain was selected since extensive historical control data were available for Wistar rats.

Test material

Test material information

1

Reference substance name:

(1E)-1-(2,6,6-trimethylcyclohex-2-en-1-yl)pent-1-en-3-one; (3E)-3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one

EC Number:

942-741-0

Cas Number:

1335-46-2

Molecular formula:

C14H22O

IUPAC Name:

(1E)-1-(2,6,6-trimethylcyclohex-2-en-1-yl)pent-1-en-3-one; (3E)-3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of tst substance (used in the report): Methylionon 70
- Chemical Identity: Mixture of isomeres: 60-70% alpha-iso-methylionone, 17-20% alpha-n-methylionone, 1-10% beta-n-methylionone, 0-5% beta-iso-methylionone
- Batch number of test material: 00119477L0

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: under N2, room temperature
- Stability of the test material:
Expiry date: 15 Jun 2022
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor and the sponsor holds this responsibility.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at start of the administration period: about 35 days
- Weight at study initiation: weight variation did not exceed 20 percent of the mean weight of each sex (P0)
- Fasting period before study: no
- Housing: up to 5 animals per sex and cage
From delivery to randomization, during overnight matings (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually. Dams and their litters were housed together until PND 21/22.
- Diet: ad libitum; mouse and rat maintenance diet “GLP” (Granovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum; tap water in water bottles
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 Jan 2021 (TS administration) To: 12 Oct 2021 (sacrifice of F1 cohort 1B animals)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 10 mg/ 100mL Cremophor)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0.4, 1.5, 6.0 g/100 mL in low, mid and high dose, respectively
- Amount of vehicle (if gavage): 10 mL/kg bw/d

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in 0.5% CMC suspension indeionized water (with 10 mg/ 100 mL Cremophor) over a period of a maximum of 7 days at room temperature were verified prior to the start of the study in a comparable batch.

At the beginning (during premating) of the administration 3 samples each were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time point additionally one sample from the mid concentration was taken for concentration control analysis. Once during the middle and once during the end of the administration each 1 sample was taken from the low, mid and high concentration for a concentration control analysis.

Duration of treatment / exposure:

F0 males: 10 weeks (premating) + 2 weeks (mating) + max. 6 weeks (post-mating)
F0 females: 10 weeks (premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 males (Co 1B): 10 weeks (post-weaning/premating) + 2 weeks (mating) + 4 weeks (post-mating)
F1 males (Co 1B): 10 weeks (post-weaning/premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 animals (Co 1A): post weaning until an approx. age of 13 weeks

Frequency of treatment:

once daily

Details on study schedule:

F0 parental animals:
After a minimum of 10 weeks after the beginning of treatment, males and females from the same dose group were mated. The females were allowed to deliver and rear their pups (F1 generation pups) until PND 4 (standardization) or PND 21 or 22 (depending on the cohort). Pups of the F1 litter were selected (F1 rearing animals) and assigned to Cohorts 1A and 1B. Before weaning of the F1 pups the F0 generation parental male animals were sacrificed. After weaning of F1 pups the F0 generation parental female animals were sacrificed.

F1 (rearing animals):
Before weaning of the F1 generation pups on PND 21, 45 male and 45 females per group were randomly selected, to be placed into cohorts. Obvious runts (those pups whose body weight was >=25% below the mean body weight of the control group, separate for sexes) were not included:
- Cohort 1A: One male and one female/litter (20/sex/group)
- Cohort 1B: One male and one female/litter (25/sex/group)
Selected F1 offspring received the test substance daily by gavage until one day before sacrifice.

F1 parental animals (Cohort 1B):
After weaning, 25 male and 25 female F1 pups per test group became F1 generation parental animals. These animals were chosen by lot and each litter was taken into account as far as technically feasible. If fewer than 25 litters were available in a group or if one sex was missing in a litter, more animals were taken from the other litters of the respective test group to obtain the required number of animals to be paired. All selected animals were treated with the test substance at the same dose level as their parents, from post-weaning through adulthood. After a minimum of 10 weeks after assignment of the F1 generation parental animals, the males and females were mated, overnight. The partners were randomly assigned, mating of siblings was avoided. The females were allowed to deliver and rear their pups (F2 generation pups) until PND 4 (standardization) or PND 21. Before weaning of the F2 pups the F1 generation parental male animals were sacrificed. The F1 generation parental females were sacrificed, shortly after the F2 generation pups had been weaned.

Standardization of litters (F1 and F2 generation pups):
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 5 male and 5 female pups (always the first 5 pups/sex and litter were taken for further rearing). If individual litters did not have 5 pups/sex, the litters were processed in such a way that the most evenly distributed 10 pups per litter were present for further rearing (e.g. 6 male and 4 female pups). Surplus animals were sacrificed. Standardization of litters was not performed in litters with <= 10 pups.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

F0: 25 animals per sex per dose
F1A: 20 animals per sex per dose
F1B: 25 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on range-finding study
- Fasting period before blood sampling for clinical biochemistry: yes, for about 16-20 hours

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality: twice daily on working days or once daily (Saturday, Sunday or on public holidays).
Cage side observations: at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
F0: animals once before the administration and subsequently once per week
F1 (Cohort 1A/1B): at weekly intervals during the administration period.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week, except
• during the mating period of the F0 and F1 parental animals, the females were weighed on the day of positive evidence of sperm GD 0 and on GD 7, 14 and 20.
• F0 and F1 females with litter were weighed on the day of parturition PND 0 and on PND 1, 4, 7, 10, 14, 18 and 21.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Generally, food consumption was determined once a week (over a period of 7 days) for male and female F0 and F1 parental animals and F1 rearing animals, with the following exceptions:
• not determined after the 2nd premating week (male F0 animals) and after the 10th premating week (male F1 animals).
• determined with evidence of sperm of the F0 and F1 females for GD 0-7, 7-14 and 14-20.
• detemined for the F0 and F1 females, which gave birth to a litter for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.

WATER CONSUMPTION: Yes
- Time schedule for examinations: once a week for the male and female F0 and F1 parental animals and F1 rearing animals, with the following exceptions:
• not determined after the 10th premating week (male F1 parental animals) and during the mating period (male and female F0 parental animals)
• determined with evidence of sperm of the F0 and F1 females for GD 0-1, 3-4, 7-8, 10-11, 14-15, 17-18 and 19-20.
• determined for F0 and F1 females, which gave birth to a litter for PND 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period and in the females after weaning.

CLINICAL PATHOLOGY
Samples were withdrawn from 10 F0 parental males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined.

Hematology
Parameters examined:
- Leukocyte count (WBC)
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)
- Prothrombin time (Hepato Quick’s test; HQT)

Clinical chemistry
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
-Cholesterol (CHOL)

Hormones:
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits. T4 was examined via ELISA.

Urinalysis
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity (SP.GR.)
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)

Oestrous cyclicity (parental animals):

evaluated by daily analysis of vaginal smear for all F0 female parental rats for all F1 female parental rats (= cohort 1B) for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.
In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female and cohort 1A and 1B female with scheduled sacrifice.

Sperm parameters (parental animals):

After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals sacrificed on schedule:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
- Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) and the F1 parents (F2 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

- Pup viability/mortality:
twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21.

- Sex ratio
determined on the day of birth (PND 0) by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.

- Anogenital distance
Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.

- Pup clinical observations
examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

- Nipple/areola anlagen
examined and counted in all surviving F1 and F2 male pups on PND 13 and on PND 20.

- Pup body weight data
on the day after birth (PND 1) and on PND 4 (before standardization), 7, 14 and 21.

- Vaginal opening
All female F1 pups selected to become the F1 parental generation females (cohort 1B) and F1 rearing animals (cohort 1A) were evaluated daily for vaginal patency beginning on PND 27. On the day of vaginal opening the body weights of the respective animals were determined.

- Preputial separation
All male F1 pups selected to become the F1 parental generation females (cohort 1B) and F1 rearing animals (chort 1A) were evaluated daily for preputial separation beginning on PND 38. On the day of preputial separation the body weights of the respective animals were determined.


CLINICAL PATHOLOGY (Cohort 1A):
Samples were withdrawn from 10 cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined.
Hematology
Parameters examined:
- Leukocyte count (WBC)
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)
- Prothrombin time (Hepato Quick’s test; HQT)

Clinical chemistry
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
-Cholesterol (CHOL)

Urinalysis
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)

Hormone analysis (F1; PND 4 and PND 22)
Blood samples were withdrawn from 10 surplus (culled) PND 4 offspring (as far as possible of different litters) per sex and group. PND 4 samples were pooled per sex and litter if the available amount is not sufficient for a hormone analysis. Blood samples were withdrawn from 10 surplus PND 22 offspring (as far as possible of different litters) per sex and group. The blood samples were collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)

Hormone analysis (F1; Cohort 1A):
Samples were withdrawn from 10 cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits. T4 was examined via ELISA.


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
All F0 generation parental animals were sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland (fixed)
11. Prostate (ventral and dorsolateral part together, fixed)
12. Testes
13. Seminal vesicles including coagulating glands (fixed)
14. Spleen
15. Thymus (fixed)
16. Thyroid glands (with parathyroid glands) (fixed)
17. Uterus with cervix
All paired organs were weighed together (left and right).


HISTOPATHOLOGY
The organs or tissues were fixed in 4% neutral-buffered formaldehyde solution except for epididymis, testis, eyes and ovaries (fixed in modified Davidson’s solution):
Organs/ tissues examined:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left
11. Esophagus
12. Eyes with optic nerve
13. Heart
14. Ileum
15. Jejunum
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Peyer’s patches
28. Rectum
29. Sciatic nerve
30. Seminal vesicles
31. Skeletal muscle
32. Spinal cord (cervical, thoracic, lumbar)
33. Spleen
34. Stomach (forestomach and glandular stomach)
35. Testis, left
36. Thymus
37. Thyroid glands
38. Trachea
39. Urinary bladder
40. Uterus
41. Vagina
42. Vas deferens

The uteri of all cohabited female F0 generation parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method [Salewski, 1964]). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl).

Postmortem examinations (offspring):

SPERM PARAMETERS (Cohort 1A)
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male cohort 1A animals sacrificed on schedule:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)

DOFC (Cohort 1A)
A differential ovarian follicle count (DOFC) was conducted in control and high dose test groups according to Plowchalk et.al. (1993). In general, sections were prepared with 2 - 3 µm thickness and serial sections were taken every 100 µm to complete about 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, H&E stained slides were prepared from all cut levels.


PATHOLOGY (F1 pups)
On PND 4, as a result of standardization, all surplus F1 pups were sacrificed by decapitation under isoflurane anesthesia and blood was sampled for determination of serum thyroid hormone concentrations. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.

On PND 22, the surplus F1 generation pups that were not used for the formation of the cohorts or any investigations were sacrificed under isoflurane anesthesia with CO2 and were examined for pathology. The selected pups for hormone analyses were sacrificed by decapitation under isoflurane anesthesia blood was sampled for thyroid hormone analyses. All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

- Organ weights (surplus F1 pups; PND 22)
The following weights were determined in up to 10 animals per sex per group sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Spleen
4. Thymus (fixed)


PATHOLOGY (Cohort 1A)
All F1 generation rearing animals, cohort 1A were sacrificed by decapitation under isoflurane anesthesia.

- Gross neropsy (Cohort 1A)
The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.

- Organ weights (Cohort 1A)
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

- Histopathology (Cohort 1A)
The organs or tissues were fixed in 4% neutral-buffered formaldehyde solution except for epididymis, testis, eyes and ovaries (fixed in modified Davidson’s solution):
Organs/ tissues examined:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left
11. Esophagus
12. Eyes with optic nerve
13. Heart
14. Ileum
15. Jejunum
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Peyer’s patches
28. Rectum
29. Sciatic nerve
30. Seminal vesicles
31. Skeletal muscle
32. Spinal cord (cervical, thoracic, lumbar)

33. Spleen
34. Stomach (forestomach and glandular stomach)
35. Testis, left
36. Thymus
37. Thyroid glands
38. Trachea
39. Urinary bladder
40. Uterus
41. Vagina
42. Vas deferens


PATHOLOGY (Cohort 1B)
All cohort 1B were sacrificed by decapitation under isoflurane anesthesia.

- Gross neropsy (Cohort 1B)
The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.

- Organ weights (Cohort 1B)
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Cauda epididymis
4. Epididymides
5. Liver
6. Ovaries
7. Pituitary gland (fixed)
8. Prostate (ventral and dorsolateral part together, fixed)
9. Seminal vesicles including coagulating gland (fixed)
10. Testes
11. Uterus (with cervix)
All paired organs were weighed together (left and right).

- Histopathology (Cohort 1B)
The organs or tissues were fixed in 4% neutral-buffered formaldehyde solution except for epididymis, testis and ovaries (fixed in modified Davidson’s solution):
Organs/ tissues examined:
1. All gross lesions
2. Adrenal glands
3. Cervix uteri
4. Coagulating glands
5. Epididymides, left
6. Liver
7. Ovaries
8. Prostate
9. Pituitary gland
10. Seminal vesicles
11. Testes, left
12. Uterus
13. Vagina

The uteri of all cohabited female cohort 1B animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method [Salewski, 1964]). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl).

- Splenic lymphocyte subpopulation analysis
10 males and 10 females of cohort 1A were used to perform a splenic lymphocyte subpopulation analysis using one half of the spleen.
The following parameters were analysed:
- B lymphocytes (B_SPL)
- T lymphocytes (T_SPL)
- CD4+ lymphocytes (CD4_SPL)
- CD8+ lymphocytes (CD8_SPL)
- Natural killer cells (NK_SPL)


PATHOLOGY (F2 pups)
After a similar standardization on PND 4, the surplus F2 pups were sacrificed under isoflurane anesthesia with CO2.
On PND 21, all F2 generation pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.

- Pup organ weights (F2 pups)
After the scheduled sacrifice, the brain, spleen and thymus of 1 pup/sex per F2 litter were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. The corresponding in-life pup weights determined on PND 21 were used to calculate the relative organ weights.

Statistics:

- DUNNETT test (two-sided): Water consumption (parental and rearing animals), food consumption (parental and rearing animals), body weight and body weight change (parental animals, rearing animals and pups; for the pup weights, the litter means were used), gestation days, duration of sexual maturation (days to vaginal opening, days to preputial separation), anogenital distance, anogenital index.
- FISHER'S EXACT test (one-sided): Male and female mating indices, male and female fertility indices, gestation index, females mated, females pregnant, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
- WILCOXON-test (one-sided): Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity)
- WILCOXON-test (one-sided) with BONFERRONI-HOLM adjustment: Spermanalysis parameters
- WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal Loss, nipple development
- WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment: Implantation sites, pups delivered, pups liveborn, live pups day x, viability index, lactation index
- WILCOXON-test (one-sided-): DOFC (differential ovarian follicular count); Follicles primordial, growing, primordial + growing
- WILCOXON test (two-sided): % live male day x, % live female day x
- KRUSKAL-WALLIS test (two-sided) + WILCOXON-test (two-sided): Number of cycles and Cycle Length, pup organ weights (absolute and relative), Organ weight parameters
- KRUSKAL-WALLIS test + WILCOXON-test (one or two-sided): Blood parameters, Urine pH, volume and specific gravity; splenic lymphocytes subpopulations

Reproductive indices:

Male reproduction data for F1 and F2 litters:
Male mating index (%) = number of males with confirmed mating / number of males placed with females x 100
Male fertility index (%) = number of males proving their fertility / number of males placed with females x 100

Female reproduction and delivery data for F1 and F2 litters:
Female mating index (%) = number of females mated / number of females placed with males x 100
Female fertility index (%) = number of females pregnant / number of females mated x 100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100

Offspring viability indices:

Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100
Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations x 100
Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth x 100
Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth x 100

*before standardization of litters (i.e. before culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males (day 0-113)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): salivation (25/25); plough nose-first into bedding (1/25)
- Test group 03 (600 mg/kg bw): salivation (25/25); unsteady gait (7/25); semiclosed eyelid (1/25); poor general condition, labored respiration and pilorection (2/25); plough nose-first into bedding (23/25)

Females (premating day 0-75)
- Test group 01 (40 mg/kg bw): salivation (3/25); eye: semiclosed eyelid, opacity and injury (1/25)
- Test group 02 (150 mg/kg bw): salivation (22/25); plough nose-first into bedding (1/25)
- Test group 03 (600 mg/kg bw): salivation (25/25); unsteady gait (18/25); semiclosed eyelid (2/25); pilorection (1/25); plough nose-first into bedding (21/25); reduced attention (1/25)
Females (mating day 0-14)
- Test group 01 (40 mg/kg bw): salivation (1/19)
- Test group 02 (150 mg/kg bw): salivation (9/20)
- Test group 03 (600 mg/kg bw): salivation (11/17); plough nose-first into bedding (3/17)
Females (gestation day 0-57)
- Test group 01 (40 mg/kg bw): saliation (5/24); eye injury, opacity, semiclosed eyelid and small eye (1/24)
- Test group 02 (150 mg/kg bw): salivation (20/25); plough nose-first into bedding (3/25)
- Test group 03 (600 mg/kg bw): salivation (23/24); plough nose-first into bedding (15/24)

- Observed salivation partly accompanied by plough nose-first into bedding in male and female animals was temporary and disappeared after a maximum of five hours which is why the effect was regarded to be test substance-induced but not adverse-

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Males (day 0-113)
- Test group 01 (40 mg/kg bw): no deaths
- Test group 02 (150 mg/kg bw): no deaths
- Test group 03 (600 mg/kg bw): sacrificed moribund (2/25) due to poor general conditions

- the mortalities in the test group 03 were assessed to be not related to the test substance as the findings (mixed cell inflammation of pleura and pericardium, inflammatory cell infiltrates in the esophagus, inflammatory process in the thoracic cavity) are suspicious of a gavage error

Females (premating day 0-75, mating day 0-14, gestation day 0-57)
- Test group 01 (40 mg/kg bw/: no deaths
- Test group 02 (150 mg/kg bw): no deaths
- Test group 03 (600 mg/kg bw): no deaths

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males (day 0-113)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): body weight - no effects; body weight change – reduced from day 42-49 (-17.3%)
- Test group 03 (600 mg/kg bw): body weight - reduced from day 49-105 (max. -9.5% on day 105); body weight change - reduced from day 35-70 and day 98-105 (max. -38.6% on day 56)
- Observed effects on body weight and body weight change in male animals was considered test item-related but not adverse

Females (premating day 0-75)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects
Females (mating day 0-14)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects
Females (gestation day 0-56)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects
Females (lactation day 0-35)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): body weight - no effect; body weight change - increased from PND 0-1 and PND 7-10 (1.5% on PND 1; 13% on PND 10), decreased from PND 4-7 (-1.2%)
- Test group 03 (600 mg/kg bw): body weight - no effect; body weight change - increased from PND0-1
- Observed effects on body weight change in female animals was considered incidental in nature, since there was no dose-dependency

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males (day 0-113)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects

Females (premating day 0-75, gestation 0-56, lactation 0-35)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Males (day 0-113)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): compared to control up to +25.0% (day 7-70)

Females (premating day 0-75)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): compared to control up to +24% (day 7-63)
Females (gestation day 0-56)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): compared to control up to +24% (GD 0-1) and +20% (GD19-20)
- Test group 03 (600 mg/kg bw): compared to control up to +41% (GD 1-20)
Females (lactation day 0-35)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects

- Observed increase in water consumption was regarded to be test substance-induced but not adverse

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): increased reticulocytes (138.6 giga/L)
- Test group 03 (600 mg/kg bw): shortened prothrombin time (32.0 sec), increased platelets count (771 giga/L), decreased hemoglobin (8.6 mmol/L), increased reticulocytes (171.1 giga/L), decreased absolute and relative eosinophil counts (0.10 giga/L; 2.0%)
- Observed effect on reticulocytes in male animals was assessed to be adaptive since a hypothetical decrease of the red blood cell parameters may be compensated which is confirmed by histological findings of extramedullary hematopoiesis in the spleens
- Observed effects on hemoglobin in male animals were within historical control range

Females
- Test group 01 (40 mg/kg bw): increased white blood cell count (2.99 giga/L), increased absolute lymphocyte count (2.15 giga/L)
- Test group 02 (150 mg/kg bw): shortened prothrombin time (33.0 sec), increased white blood cell count (2.88 giga/L)
- Test group 03 (600 mg/kg bw): shortened prothrombin time (31.7 sec), increased white blood cell count (3.80 giga/L), increased absolute lymphocyte count (2.73 giga/L), increased absolute large unstained cell counts (0.02 giga/L), decreased relative basophil count (0.3%)

- Observed effects on prothrombin time and platelets count in male and female animals were assessed to be test item-related and adverse
- Observed effects related to white blood cell count in male and female animals were within historical control range

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males
- Test group 01 (40 mg/kg bw): decreased alanine aminotransferase (ALT) activities (0.64 µkat/L)
- Test group 02 (150 mg/kg bw): increased total protein (66.69 g/L), increased globulin (30.88 g/L), increased calcium levels (2.65 mmol/L), decreased total bilirubin level (1.15 µmol/L)
- Test group 03 (600 mg/kg bw): increased γ-glutamyl transferase (GGT) activities (69 nkat/L), increased total protein (70.04 g/L), increased globulin (38.02 g/L), increased calcium levels (2.67 mmol/L), decreased glucose levels (3.88 mmol/L), decreased alanine aminotransferase (ALT) activities (0.58 µkat/L), increased albumin (38.02 g/L) and potassium levels (5.00 mmol/L), decreased chloride levels (101. mmol/L)

Females
- Test group 01 (40 mg/kg bw): increased urea levels, decreased inorganic phosphate level
- Test group 02 (150 mg/kg bw): increased urea levels, increased sodium levels, decreased total bilirubin level
- Test group 03 (600 mg/kg bw): increased γ-glutamyl transferase (GGT) activities (40 nkat/L), increased total protein, increased globulin, increased calcium levels, decreased glucose levels, increased triglyceride values, decreased alkaline phosphatase (ALP) activities (0.58 µkat/L), decreased chloride levels, increased urea levels, increased albumin levels, increased sodium levels, decreased total bilirubin level

- Observed effects on total protein (male/female animals), globulin (male/female animals), calcium levels (male/female animals), GGT (male/female animals), glucose (male/female animals) and triglyceride (female animals) values were regarded as treatment-related and adverse
- Observed effects on ALT (male animals) and ALP (female animals) were regarded as treatment-related and adaptive (PSD Guidance Document: Toxicological Significance of Reduced Levels of Serum ALT and/or AST in Animal Studies, May 2007)
- Observed effects on albumin (male/female animals), potassium (male animals), chloride (male/female animals), urea (female animals) and sodium levels (female animals) were within historical control ranges
- Observed effects on total bilirubin level (male/female animals) and inorganic phosphate level (female animals) were regarded as incidental since these changes were not dose-dependent

Endocrine findings:

not specified

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): decreased pH (6.2)
- Test group 03 (600 mg/kg bw): increased ketone body levels (2.0), decreased pH (6.2), increased incidences of granular and epithelial casts in urine sediment (0.8), increased urine volume (9.1 mL)
- Observed effects on ketone body levels and pH in male animals was regarded as treatment-related and adverse
- Observed effect on incidences of granular and epithelial casts in male animals was regarded as not human relevant as this is a finding typically observed in male rats of this age class in combination with an alpha-2u globulinuria which was confirmed histopathologically (Hard, G. C. Mechanisms of Rodent Renal Carcinogenesis Revisited. Toxicologic Pathology, 46 (8), 956-969 (2018))
- Observed effects on urine volume and urine specific gravity in male animals were assessed as adaptive mechanism of the kidneys to a changed fluid income

Females
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): decreased pH (5.3), increased urine specific gravity (1,054 g/L)
- Observed effects on urine specific gravity as well as change in urine pH were assessed as adaptive mechanism of the kidneys to a changed fluid income and acid-base homeostasis in blood, respectively 

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Male
Bile:
- Test group 03 showed an increased incidence and/or severity of diffuse bile duct hyperplasia => regarded as treatment-related
Liver:
- Single male animals of test groups 01 to 03 showed a minimal, diffuse hepatocellular hypertrophy with a minimally increased incidence in test groups 02 and 03. Although, a clear dose response relationship was not recognized. => regarded as treatment-related (in combination with increased liver weights)
Thyroid glands:
- test groups 02 and 03 showed a minimal to moderate diffuse follicular hypertrophy and hyperplasia => regarded as treatment-related (in combination with increased relative thyroid gland weights)
- test group 02 and 03 showed an increased amount of altered colloid in thyroid glands => regarded as treatment-related (in combination with increased relative thyroid gland weights)
Kidneys:
- test group 03 showed an increased incidence and severity of eosinophilic, intracytoplasmic droplets in proximal convoluted tubules, that was associated to granular casts in tubules at the junction of the outer and inner stripes of the outer medulla and an increased incidence and severity of basophilic tubules; in individual animals, eosinophilic droplets were exemplarily stained with an antibody against alpha 2u-Globulin and the globules were positive for alpha 2u-Globulin in all respective animals - therefore, the presence of alpha 2u-Globulin nephropathy is likely => regarded as treatment-related but not relevant for humans
- test group 02 showed a minimally increased severity of basophilic tubules and minimal granular casts in 3/20 animals => regarded as treatment-related (in combination with increased kidney weights)
Spleen:
- test group 02 and 03 showed an increased incidence and/or severity of extramedullary hematopoiesis => regarded as treatment-related (in combination with increased numbers of erythropoietic precursor cells)

Female
Bile:
- Test group 03 showed an increased incidence and/or severity of diffuse bile duct hyperplasia => regarded as treatment-related
Liver:
- Test groups 02 and 03 presented a minimal up to moderate centrilobular hepatocellular hypertrophy => regarded as treatment-related (in combination with increased absolute and relative liver weights)
Thyroid glands:
- test groups 02 and 03 showed a minimal to moderate diffuse follicular hypertrophy and hyperplasia => regarded as treatment-related (in combination with increased relative thyroid gland weights)
- test group 02 and 03 showed an increased amount of altered colloid in thyroid glands => regarded as treatment-related (in combination with increased absolute and relative thyroid gland weights)

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Hormones:
Males
- Test group 01 (40 mg/kg bw): decreased T4 value (60.44 nmol/L), increased TSH value (not significant)
- Test group 02 (150 mg/kg bw): decreased T4 value (60.62 nmol/L)
- Test group 03 (600 mg/kg bw): decreased T4 value (48.34 nmol/L), increased TSH value (not significant)
- Observed effects on TSH and T4 in male animals were assessed as treatment-related and adverse in the test group 03 due to histopathological findings in the thyroids and increased relative thyroid weights
- Observed effect on T4 in male animals of test group 01 and 02 was regarded as maybe treatment related but not-adverse

Females
- Test group 01 (40 mg/kg bw): decreased TSH value (4.43 µg/L)
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): increased TSH value (7.82 µg/L)
- Observed effect on TSH in female animals of test group 01 were within historical control range and regarded as incidental and not treatment related
- Observed effect on TSH in female animals of test group 03 were assessed as treatment-related and adverse due to histopathological findings in the thyroids and increased absolute and relative thyroid weights

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Females (last 3 weeks prior to mating)
- Test group 01 (40 mg/kg bw): no effects; mean 4.0
- Test group 02 (150 mg/kg bw): no effects; mean 4.1
- Test group 03 (600 mg/kg bw): no effects; mean 4.0

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Males
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects

Reproductive performance:

no effects observed

Description (incidence and severity):

Male
Mating Index:
- Test group 01 (40 mg/kg bw): 96%
- Test group 02 (150 mg/kg bw): 100%
- Test group 03 (600 mg/kg bw): 100%
Fertility index:
- Test group 01 (40 mg/kg bw): 92%
- Test group 02 (150 mg/kg bw): 100%
- Test group 03 (600 mg/kg bw): 100%
These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
Histopathological findings:
- Test group 01 (40 mg/kg bw): one animal showed a reduced size of both testes and epididymides, that correlated to a diffuse massive degeneration/atrophy and to a massive oligospermia in testes and epididymides, respectively
- Test group 02 (150 mg/kg bw): no
- Test group 03 (600 mg/kg bw): no

Females
Mating index:
- Test group 01 (40 mg/kg bw): 96%
- Test group 02 (150 mg/kg bw): 100%
- Test group 03 (600 mg/kg bw): 96%
Mean duration until sperm was detected:
- Test group 01 (40 mg/kg bw): 2.3 days
- Test group 02 (150 mg/kg bw): 2.2 days
- Test group 03 (600 mg/kg bw): 2.2 days
All femals rats delivered pups or had implants in utero with the following exception:
- Test group 01 (40 mg/kg bw): 2 animals did not become pregnant.
- Test group 03 (600 mg/kg bw): 1 animal did not become pregnant.
Fertility index:
- Test group 01 (40 mg/kg bw): 96%
- Test group 02 (150 mg/kg bw): 100%
- Test group 03 (600 mg/kg bw): 100%
These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
Histopathological findings:
- Test group 01 (40 mg/kg bw): no
- Test group 02 (150 mg/kg bw): no
- Test group 03 (600 mg/kg bw): no
Duration of gestation:
- Test group 01 (40 mg/kg bw): 22.2 days
- Test group 02 (150 mg/kg bw): 22.2 days
- Test group 03 (600 mg/kg bw): 22.2 days
Gestation index:
- Test group 01 (40 mg/kg bw): 100%
- Test group 02 (150 mg/kg bw): 96%
- Test group 03 (600 mg/kg bw): 100%
Implantation:
- Test group 01 (40 mg/kg bw): 12.8 implants/dam
- Test group 02 (150 mg/kg bw): 12.5 implants/dam
- Test group 03 (600 mg/kg bw): 12.0 implants/dam
Postimplantation loss:
- Test group 01 (40 mg/kg bw): 8.3 mean %
- Test group 02 (150 mg/kg bw): 10.2 mean %
- Test group 03 (600 mg/kg bw): 6.6 mean %
Mean number of pups:
- Test group 01 (40 mg/kg bw): 11.8 pups/dam
- Test group 02 (150 mg/kg bw): 11.4 pups/dam
- Test group 03 (600 mg/kg bw): 11.3 pups/dam
Rate of liveborn pups
- Test group 01 (40 mg/kg bw): 99%
- Test group 02 (150 mg/kg bw): 99%
- Test group 03 (600 mg/kg bw): 99%

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity F0

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical pathology and histopathological findings observed at 150 mg/kg bw/d

2

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity, F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical pathology and histopathological findings observed at 600 mg/kg bw/d

3

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male (day 0-65)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): salivation (16/20), plough nose-first into bedding (1/20)
Test group 13 (600 mg/kg bw): salivation (20/20), plough nose-first into bedding (19/20)

Female (day 0-65)
Test group 11 (40 mg/kg bw): salivation (2/20)
Test group 12 (150 mg/kg bw): salivation (18/20), plough nose-first into bedding (1/20), general poor condition + labored respiration + piloerection (1/20)
Test group 13 (600 mg/kg bw): salivation (20/20), plough nose-first into bedding (20/20)

- observed effect of salivation in male and female animals was reversible within in a maximum of two hours after administration and, thus, was regarded as treatment related but not adverse



Cohort F1B
Male (day 0-94)
Test group 11 (40 mg/kg bw): head injury
Test group 12 (150 mg/kg bw): salivation (23/25), plough nose-first into bedding (1/25),
Test group 13 (600 mg/kg bw): salivation (22/25); plough nose-first into bedding (20/25); piloerection, labored respiration, poor general conditions (1/25)

Female (day 0-75)
Test group 11 (40 mg/kg bw): semiclosed eyelid, closed eyelid, pale skin, poor general condition, hard abdomen, abdominal position, palpable in abdomen (1/25 – was sacrificed moribund)
Test group 12 (150 mg/kg bw): salivation (21/25), plough nose-first into bedding (3/25)
Test group 13 (600 mg/kg bw): salivation (25/25), plough nose-first into bedding (23/25)
Female (mating)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): salivation (3/14)
Test group 13 (600 mg/kg bw): salivation (9/14)
Female (gestation)
Test group 11 (40 mg/kg bw): salivation (1/24)
Test group 12 (150 mg/kg bw): salivation (23/25)
Test group 13 (600 mg/kg bw): salivation (23/25), plough nose-first into bedding (21/25)
Female (lactation)
Test group 11 (40 mg/kg bw): salivation (6/22)
Test group 12 (150 mg/kg bw): salivation (22/25), supernumerary nipple (1/23)
Test group 13 (600 mg/kg bw): salivation (24/24), plough nose-first into bedding (23/24)

- salivation and accompanied plough nose-first into bedding temporary and were regarded test-item related but not adverse
- all other observed events were regarded to be not related to test item administration

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Cohort F1A
Male
Test group 11 (40 mg/kg bw): no deaths
Test group 12 (150 mg/kg bw): no deaths
Test group 13 (600 mg/kg bw): no deaths

Female
Test group 11 (40 mg/kg bw): 1 accidental death
Test group 12 (150 mg/kg bw): 1 death after poor general condition
Test group 13 (600 mg/kg bw): no deaths
- observed mortalities in female animals was regarded as incidental and not test item related



Cohort F1B
Male
Test group 11 (40 mg/kg bw): no deaths
Test group 12 (150 mg/kg bw): no deaths
Test group 13 (600 mg/kg bw): no deaths

Female
Test group 11 (40 mg/kg bw): 1 animal showed a palpable mass in the abdomen, poor general state, paleness, semiclosed/closed eyelids, hard abdomen and abdominal position from inlife day 53 onwards and was sacrificed moribund on day 57. Histopathology indicated an agonal heart failure.
Test group 12 (150 mg/kg bw): no deaths
Test group 13 (600 mg/kg bw): no deaths
- observed event in single female of test group 11 was regarded as incidental

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male (day 0-56)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): significantly reduced throughout total study duration with a maximum of -15.2% at day 63
- observed effect in male animals was regarded as treatment-related

Female (day 0-56)
Test group 11 (40 mg/kg bw): body weight change increased at day 56-63 (11.7+/-6.4)
Test group 12 (150 mg/kg bw): -7.2% at day 0
Test group 13 (600 mg/kg bw): -13.5% at day 0, -7.9% at day 7
- observed effect in female animals of test group 11 was regarded as incidental
- observed effect in female animals of test group 12 and 13 was regarded as test item-related



Cohort F1B
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): significantly reduced throughout d0-7 and d35-91 (maximum decrease of -10.5% on d77); body weight change of high-dose males was statistically reduced from inlife days 35-63, 70-77 and at the total interval days 0-91
- observed effect in male animals was regarded as treatment-related

Female
Test group 11 (40 mg/kg bw): decreased body weight change during d35-42
Test group 12 (150 mg/kg bw): increased body weight change during d21-28
Test group 13 (600 mg/kg bw): -8.8% at day 0, -5.8% at day 7
- observed effect in female animals of test group 11 and 12 were regarded as incidental
- observed effect in female animals of test group 13 was regarded as test item-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Cohort F1A
Male (day 0-56)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects

Female (day 0-56)
Test group 11 (40 mg/kg bw): +8.9% at day 42-49
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
- observed effect in female animals was regarded as incidental



Cohort F1AB
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects

Female
Test group 11 (40 mg/kg bw): -9.0% at day 21-28
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
- observed effect in female animals was regarded as incidental

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male (day 0-56)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): +10.9% at day 10-14 and +12.6% at day 52-56
- observed effect in male animals was regarded as treatment-related

Female (day 0-56)
Test group 11 (40 mg/kg bw): +15.5% at day 52-56
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): +12.8% at day 10-14; +11.2% at day 17-21
- observed effect in female animals was regarded as unrelated to test item administration as no dose-dependency could be established



Cohort F1B
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects

Female (pre-mating)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
Female (gestation)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): increased on GD 10-11 (+18%) and 17-18 (+19%)
Test group 13 (600 mg/kg bw): increased during GD 7 to 21 (up to 44% on GD 21)
Female (lactation)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): increased on PND 1-2 (+16%)
Test group 13 (600 mg/kg bw): increased on PND 1-2 (+24%) and 7-8 (+15%)
- observed effect in female animals was regarded as related to the test item

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
No treatment-related, adverse changes among hematological parameters were observed
Male
Test group 11 (40 mg/kg bw): +20.12% white blood cell count
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): +23.4% reticulocytes
- observed effect on reticulocytes was regarded as adaptive/compensatory due to absence of any signs of anemia and histopathological findings (extramedullary hematopoiesis in the spleen)

Female
Test group 11 (40 mg/kg bw): +25.18% absolute lymphocyte count, +41.54% absolute monocyte count, +100% absolute basophile count, +60.0% basophile count
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): +33.32% absolute lymphocyte count, +44.64% absolute monocyte count
- observed effect on white blood cell count was regarded as maybe test item-related but non-adverse since this is the only relevantly changed differential blood cell fraction (ECETOC Technical Report No. 85, 2002)

- all other effects are regarded as incidental and not treatment-related as no dose-dependency could be established



Not performed in Cohort F1B

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male
Test group 11 (40 mg/kg bw): decreased alanine aminotransferase (ALT)
Test group 12 (150 mg/kg bw): decreased bilirubin; increased total protein and albumin; increased calcium
Test group 13 (600 mg/kg bw): increased γ-glutamyl transferase (GGT); increased total protein, albumin and globulin; decreased glucose; decrease in alanine aminotransferase (ALT); decreased bilirubin; decreased chloride; increased calcium
- observed effect in test group 13 on GGT, total protein, albumin, globulin and glucose were regarded as treatment-related and adverse
- observed effect in test group 13 on ALT and in test group 13 and 13 on total bilirubin were regarded as treatment related but adaptive rather than adverse since it most probably occurred due to liver enzyme induction (PSD guidance, 2007) and an increased bilirubin conjugation followed by an accelerated release via the bile
- observed effects in total protein and albumin in test group 12 were regarded as non-adverse if at all treatment related (ECETOC Technical Report No. 85, 2002)
- all other findings were regarded as incidental since no dose-dependency could be established or values were within the historical control range

Female
Test group 11 (40 mg/kg bw): increased cholesterol and calcium levels
Test group 12 (150 mg/kg bw): increased urea; increased triglyceride
Test group 13 (600 mg/kg bw): no effects increased γ-glutamyl transferase (GGT); increased total protein, albumin and globulin; increased urea; decreased glucose values and increased globulin; decreased chloride
- observed effect in test group 13 on GGT, total protein, albumin and globulin were regarded as treatment-related and adverse
- all other findings were regarded as incidental since no dose-dependency could be established or values were within the historical control range



Not performed in Cohort F1B

Endocrine findings:

not specified

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male
Test group 11 (40 mg/kg bw): incidence of casts (0.6)
Test group 12 (150 mg/kg bw): incidence of transitional epithelial cells and granular and epithelial casts in urine sediment (1.7 and 1.9), increased pH (6.4)
Test group 13 (600 mg/kg bw): increased ketone body level (1.5), decreased pH (5.8), incidence of transitional epithelial cells and granular and epithelial casts in urine sediment (1.6 and 1.9), increased specific gravity (1.046 g/L)
- observed effect on ketone body level and decreased pH in test group 13 were regarded as treatment-related and adverse
- observed effect on casts was related to alpha-2u globulinuria which was confirmed histopathologically and is thus not regarded as relevant for humans (Hard et al., 2018)
- all other effects were regarded as isolated findings and as adaptation of the fluid income in the kidneys and an adaptive regulation of the acid-base homeostasis in blood

Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): increased urine volume
- observed effect on increased urine volume was regarded as isolated findings and as adaptation of the fluid income in the kidneys



Not performed in Cohort F1B

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Terminal body weight
Male: -2.4% / -1.7% / -13.6%** versus control in test group 11 / 12 / 13, respectively => regarded as treatment related

Absolute organ weight
Male:
- Adrenal gland: -0.4% / -1.0% / -14.48%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Heart: -4.4% / -5.0% / -10.7%** versus control in test group 11 / 12 / 13, respectively =>
- Kidneys: +1.9% / +7.6%* / +12.1%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Liver: -0.2% / +13.8%** / +25.4%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Thymus: -12.3% / -8.4% / -28.1%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
Female
- Liver: +4.3% / +12.9%** / +49.7%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Pituitary gland: +20.5%* / +15.%* / +8.9% versus control in test group 11 / 12 / 13, respectively => not treatment related, as there was no dose-response relationship or a correlating histopathological finding
- Thyroid glands: +4.4% / +7.0% / +26.6%** versus control in test group 11 / 12 / 13, respectively => treatment related

Relative organ weight
Male:
- Brain: +2.1% / +1.6% / +13.0%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Cauda epididymides: -1.5% / -7.3%* / +13.3%* versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Epididymides: -1.3% / -2.9% / +10.2%* versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Heart: -2.2% / -3.6% / +3.0%* versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Kidneys: +4.4% / +9.4%** / +29.8%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Liver: +2.2% / +15.6%** / +45.1%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Spleen: +4.8% / +5.5% / +21.0%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Testes: 0.0% / -1.4% / +13.7%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Thyroid glands: +.17% / +11.5%* / +22.2%** versus control in test group 11 / 12 / 13, respectively => treatment related
Female:
- Heart: -0.7% / +1.1% / +6.6%* versus control in test group 11 / 12 / 13, respectively => not treatment related (absolute weights not increased, no histopathological finding)
- Kidneys: +0.8% / +3.3%* / +10.3%** versus control in test group 11 / 12 / 13, respectively => treatment related for test group 13 (above historical control range)
- Liver: +3.2% / +13.1%** / +53.7%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Pituitary glands: +18.4%* / +14.5%* / +10.8% versus control in test group 11 / 12 / 13, respectively => not treatment related, as there was no dose-response relationship or a correlating histopathological finding
- Spleen: +2.5% / -0.8% / +13.9%** versus control in test group 11 / 12 / 13, respectively => not treatment related (absolute weights not increased, no histopathological finding)
- Thyroid glands: +4.0% / +7.1%* / +29.3%** versus control in test group 11 / 12 / 13, respectively => treatment related for test group 13 (above historical control range)

*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group.



Cohort F1B
Terminal body weight
Male: -3.0% / -1.9% / -12.0%** versus control in test group 11 / 12 / 13, respectively => regarded as treatment related

Absolute organ weight
Male:
- Liver: -2.6% / +14.8%** / +27.4%** versus control in test group 11 / 12 / 13, respectively => regarded as treatment related
Female
- Liver: +3.7% / +13.4%** / +49.4%** versus control in test group 11 / 12 / 13, respectively => regarded as treatment related
- Ovaries: +1.8% / +6.1% / +13.5%** versus control in test group 11 / 12 / 13, respectively => very unlikely to be test item related (ovaries of cohort F1A animals did not have a dose related weight increase or any histopathological change, or significant results of the DOFC)

Relative organ weight
Male:
- Cauda epididymides: +8.4%* / +4.3% / +13.8%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight for test group 13; incidental for test group 11 since no dose-response relationship
- Epididymides: +3.2% / 0.0% / +10.2%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Liver: +0.6% / +17.1%** / +44.7%** versus control in test group 11 / 12 / 13, respectively => regarded as treatment related
- Seminal Vesicles: +1.6% / +4.0% / +16.4%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Testes: +1.9% / -1.3% / +11.5%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
Female:
- Adrenal glands: +1.6% / +3.6% / +15.2%** versus control in test group 11 / 12 / 13, respectively => not treatment related (absolute weights not increased, no histopathological finding, no similar effect in cohort F1A)
- Liver: +3.1%** / +14.0%** / +55.0%** versus control in test group 11 / 12 / 13, respectively => treatment related for test group 12 and 13; regarded as not treatment related for test group 11 (only relative weight parameters were significantly changed; weight increase was only minimal; absolute and relative liver weights lay below the range of historical controls)
- Ovaries: +1.3% / +6.8% / +17.2%** versus control in test group 11 / 12 / 13, respectively => very unlikely to be test item related (ovaries of cohort F1A animals did not have a dose related weight increase or any histopathological change, or significant results of the DOFC)

*: p <= 0.05, **: p <= 0.01

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Surplus F1 generation pups on PND 22
Terminal body weight
Female: -1.1% / -2.2% / -9.0%* versus control in test group 11 / 12 / 13, respectively => regarded as treatment related

Absolute organ weights
Male:
No effects
Female:
- spleen: -3.5% / -10.8% / -15.1%* => related to the decreased terminal body weight

*: p <= 0.05, **: p <= 0.01

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): enlarged kidney (2/20), enlarged spleen (1/20)
Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



Cohort F1B
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): macroscopically visible dark brown discoloration of the liver (4/25)
Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): macroscopically visible dark brown discoloration of the liver (7/25)
- observed effect of discoloration in the liver was regarded as treatment related
- All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Surplus F1 generation pups on PND 22
Macroscopic findings were not observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male
Bile:
- Test group 13 showed an increased incidence and/or severity of diffuse bile duct hyperplasia => regarded as treatment-related
Liver:
- Test groups 02 to 03 showed a minimal up to moderate centrilubolar hepatocellular hypertrophy => regarded as treatment-related (in combination with increased liver weights)
Thyroid glands:
- few animals of test group 13 showed a minimal to slight diffuse follicular hypertrophy and hyperplasia; animals of test group 12 and 13 presented additionally an increased amount of altered colloid => regarded as treatment-related (in combination with increased relative thyroid gland weights)
Kidneys:
- test groups 12 and 13 showed an increased incidence and severity of eosinophilic, intracytoplasmic droplets in proximal convoluted tubules, that was associated to increased incidence and severity of basophilic tubules; test group 13 showed additionally slight to moderate granular casts in tubules at the junction of the outer and inner stripes of the outer medulla which were composed of proteinaceous material admixed with cellular debris; in individual animals, eosinophilic droplets were exemplarily stained with an antibody against alpha 2u-Globulin and the globules were positive for alpha 2u-Globulin in all respective animals - therefore, the presence of alpha 2u-Globulin nephropathy is likely => regarded as treatment-related but not relevant for humans
- test group 11 showed likewise an increased incidence of eosinophilic droplets in proximal convoluted tubules, and of basophilic tubules=> regarded as treatment-related (within historical control range but increased incidence compared to the control group)
Spleen:
- test group 13 showed an increased incidence and severity of extramedullary hematopoiesis => regarded as not treatment-related (no clear dose response relationship)
Thymus:
- test group 13 males showed an increased incidence of increased cellularity of epithelial cells => incidental (no effect in females, no effects in P0 animals, level below female F0 control)

Female
Bile:
- Test group 13 showed an increased incidence and/or severity of diffuse bile duct hyperplasia => regarded as treatment-related
Liver:
- Test groups 02 to 03 showed a minimal up to moderate centrilubolar hepatocellular hypertrophy => regarded as treatment-related (in combination with increased liver weights)
- Test group 13 showed an increased incidence and severity of periportal vacuolation which upon ORO-stain turned out to be lipid droplets => treatment related
Thyroid glands:
- few animals of test group 13 showed a minimal to slight diffuse follicular hypertrophy and hyperplasia => regarded as treatment-related (in combination with increased relative thyroid gland weights)
- test group 11 and 13 presented additionally an increased amount of altered colloid => not treatment related (since no dose response relationship)

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.


Differential ovarian follicle count in Cohort F1A
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and animals of test group 13.



Not performed on Cohort F1B

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Hormones
Surplus pups PND4 + PND22
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects

Cohort F1A
Male
Test group 11 (40 mg/kg bw): decreased T4 (within historical control range)
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw: decreased T4 (-34.0%; below historical control range)
- observed T4 decrease in males of test group 13 in combination with histopathologic findings in the thyroid and increased relative thyroid weights was regarded as treatment related and adverse whereas T4 decrease in males of test group 11 was regarded as incidental and not treatment related
Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): increased TSH (marginally above historical control range)
- in combination with histopathologic findings in the thyroids and increased absolute and relative thyroid weights, higher TSH values in females of test group 13 were regarded as treatment related and adverse

Lymphocyte subpopulation in spleen
Cohort F1A
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects



Hormones
Not performed in Cohort F1B

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Cohort F1A
Female (generated for 2 weeks around PND75)
Test group 11 (40 mg/kg bw): no effects / 3.9 days
Test group 12 (150 mg/kg bw): no effects / 3.9 days
Test group 13 (600 mg/kg bw): no effects / 3.9 days



Cohort F1B
Female (during the last 3 weeks prior to mating)
Test group 11 (40 mg/kg bww): 4.0 days duration / 4.4 cycles
Test group 12 (150 mg/kg bw): 4.0 days duration / 4.6 cycles
Test group 13 (600 mg/kg bw): 4.0 days duration / 4.4 cycles
- observed reduced cycle number in test group 11 and 13 were regarded as incidental (no dose-dependency, absence of corroborating effect in cohort F1A)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Cohort F1A
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects



Not examined for Cohort F1B

Reproductive performance:

no effects observed

Description (incidence and severity):

Not performed on Cohort F1A



Cohort F1B
Male
Mating Index:
- Test group 11 (40 mg/kg bw): 100%
- Test group 12 (150 mg/kg bw): 100%
- Test group 13 (600 mg/kg bw): 100%
Fertility index:
- Test group 11 (40 mg/kg bw): 96%
- Test group 12 (150 mg/kg bw): 92%
- Test group 13 (600 mg/kg bw): 92%
These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility.
Histopathological findings:
- Test group 11 (40 mg/kg bw): no
- Test group 12 (150 mg/kg bw): no
- Test group 13 (600 mg/kg bw): no

Females
Mating index:
- Test group 11 (40 mg/kg bw): 100%
- Test group 12 (150 mg/kg bw): 100%
- Test group 13 (600 mg/kg bw): 100%
Mean duration until sperm was detected:
- Test group 11 (40 mg/kg bw): 2.3 days
- Test group 12 (150 mg/kg bw): 2.3 days
- Test group 13 (600 mg/kg bw): 2.2 days
All female rats delivered pups or had implants in utero with the following exception:
- Test group 10: 1 animal did not become pregnant
- Test group 11: 2 animals did not become pregnant
- Test group 12: 2 animals did not become pregnant
- Test group 13: 1 animal did not become pregnant
Fertility index:
- Test group 11 (40 mg/kg bw): 91.7%
- Test group 12 (150 mg/kg bw): 92.0%
- Test group 13 (600 mg/kg bw): 96.0%
Histopathological findings:
- Test group 11 (40 mg/kg bw): no
- Test group 12 (150 mg/kg bw): no
- Test group 13 (600 mg/kg bw): no
Duration of gestation:
- Test group 11 (40 mg/kg bw): 22.2 days
- Test group 12 (150 mg/kg bw): 22.0 days
- Test group 13 (600 mg/kg bw): 22.2 days
Gestation index:
- Test group 11 (40 mg/kg bw): 100%
- Test group 12 (150 mg/kg bw): 100%
- Test group 13 (600 mg/kg bw): 100%
Implantation:
- Test group 11 (40 mg/kg bw): 11.5 implants/dam
- Test group 12 (150 mg/kg bw): 11.7 implants/dam
- Test group 13 (600 mg/kg bw): 12.6 implants/dam
Postimplantation loss:
- Test group 11 (40 mg/kg bw): 5.6 mean %
- Test group 12 (150 mg/kg bw): 6.1 mean %
- Test group 13 (600 mg/kg bw): 3.8 mean %
Mean number of pups:
- Test group 11 (40 mg/kg bw): 11.0 pups/dam
- Test group 12 (150 mg/kg bw): 11.0 pups/dam
- Test group 13 (600 mg/kg bw): 12.2 pups/dam
Rate of liveborn pups
- Test group 11 (40 mg/kg bw): 99.2%
- Test group 12 (150 mg/kg bw): 98.0%
- Test group 13 (600 mg/kg bw): 96.6%

Effect levels (P1)

1

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weights in males and clinical pathology and histopathological findings observed at 600 mg/kg bw/d:

2

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance F1

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Target system / organ toxicity (P1)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Test group 01 (40 mg/kg bw): no effects
Test group 02 (150 mg/kg bw): no effects
Test group 03 (600 mg/kg bw): no effects

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Number of liveborn pups / percentage of stillborn pups:
Test group 00 (00 mg/kg bw): 11.4 / 1.0%
Test group 01 (40 mg/kg bw): 11.7 / 1.1%
Test group 02 (150 mg/kg bw): 11.3 / 0.7%
Test group 03 (600 mg/kg bw): 11.2 / 0.7%
Viability index (PND 0-4):
Test group 00 (00 mg/kg bw): 97.1%
Test group 01 (40 mg/kg bw): 95.0%
Test group 02 (150 mg/kg bw): 99.7%
Test group 03 (600 mg/kg bw): 97.8%
Lactation index (PND 4-21):
Test group 00 (00 mg/kg bw): 99.3%
Test group 01 (40 mg/kg bw): 100.0%
Test group 02 (150 mg/kg bw): 100.0%
Test group 03 (600 mg/kg bw): 100.0%
- the test substance did not influence pre-weaning F1-pup survival in all test groups

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male:
Test group 01 (40 mg/kg bw): no effects
Test group 02 (150 mg/kg bw): no effects
Test group 03 (600 mg/kg bw): reduced on PND 14 (-7.6%), body weight change between PND 7-14
Female:
Test group 01 (40 mg/kg bw): no effects
Test group 02 (150 mg/kg bw): no effects
Test group 03 (600 mg/kg bw): reduced on PND 14 (-7.9%), body weight change between PND 7-14, reduced in the total interval PND 1-21

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio:
% live male pups PND 0 vs. PND 21 / % live female pups PND 0 vs. PND 21:
Test group 00 (00 mg/kg bw): 52.5 vs. 52.8 / 47.5 vs. 47.2
Test group 01 (40 mg/kg bw): 53.9 vs. 51.0 / 46.1 vs. 49.0
Test group 02 (150 mg/kg bw): 46.6 vs. 46.4 / 53.4 vs. 53.6
Test group 03 (600 mg/kg bw): 52.0 vs. 51.6 / 48.0 vs. 48.4
- no substantial difference; slight differences were regarded to be spontaneous in nature

Vaginal opening in females:
Days to reach criterion / mean body weight on that day:
Test group 00 (0 mg/kg bw): 31.6 d / 98.0 g
Test group 01 (40 mg/kg bw): 30.6 d / 91.5 g*
Test group 02 (150 mg/kg bw): 30.9 d / 91.7 g*
Test group 03 (600 mg/kg bw): 30.9 d / 87.6 g**
* p<=0.05, ** p <=0.01
- observed effect on body weights were within historical range of the strain and is, thus, considered to be not related to the compound

Preputial separation in males:
Days to reach criterion / mean body weight on that day:
Test group 00 (0 mg/kg bw): 41.2 / 173.4 g
Test group 01 (40 mg/kg bw): 41.5 d / 171.6 g
Test group 02 (150 mg/kg bw): 41.4 d / 172.4 g
Test group 03 (600 mg/kg bw): 42.2 d** / 165.8 g
** p <=0.01
- Although the time of preputial separation was slightly later in the high dose group, the animals of this dose group reached sexual maturity even at a slightly lower body weight when compared to controls. Therefore, the difference in timing of preputial separation is considered not to be a delay in commencement of sexual maturity, but due to lower postnatal body weight development. Additionally, all values for days and weights are well within the historical control range.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Males:
Test group 00 (00 mg/kg bw): 3.08 mm / 1.62 (Index Cubic Root)
Test group 01 (40 mg/kg bw): 3.12 mm / 1.65 (Index Cubic Root)
Test group 02 (150 mg/kg bw): 3.08 mm / 1.63 (Index Cubic Root)
Test group 03 (600 mg/kg bw): 3.04 mm / 1.58 (Index Cubic Root)
Females:
Test group 00 (00 mg/kg bw): 1.38 mm / 0.74 (Index Cubic Root)
Test group 01 (40 mg/kg bw): 1.41 mm / 0.76 (Index Cubic Root)
Test group 02 (150 mg/kg bw): 1.37 mm / 0.73 (Index Cubic Root)
Test group 03 (600 mg/kg bw): 1.39 mm / 0.74 (Index Cubic Root)

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Nipple development (% / number) on day 13
Test group 01 (40 mg/kg bw): 40.9% */ 1.0
Test group 02 (150 mg/kg bw): 64.1%** / 1.9*
Test group 03 (600 mg/kg bw): 59.6%** / 2.2**
* p<=0.05, ** p <=0.01
Nipple development (% / number) on day 20
Test group 01 (40 mg/kg bw): 0.0 / 0.0
Test group 02 (150 mg/kg bw): 0.0 / 0.0
Test group 03 (600 mg/kg bw): 0.0 / 0.0
- effects observed show no dose-dependency and values are within historical control range
Within this context it has to be mentioned that there was an obvious negative correlation between the litter weight on PND 14 and the number of observed nipples / areola anlagen, i.e. the litters which had the highest incidences had also quite low litter weight. Thus, the observed differences are considered rather to be a consequence of delayed body weight development than to represent an antiandrogenic action of the test compound. This is also strengthened by the absence of any nipple / areola anlagen on PND 20.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Test group 01 (40 mg/kg bw): post-mortem autolysis (2/87), general finding - not assessed (2/87)
Test group 02 (150 mg/kg bw): post-mortem autolysis (1/81), empty stomach (1/81)
Test group 03 (600 mg/kg bw): empty stomach (2/87)
Females:
Test group 01 (40 mg/kg bw): general finding - not assessed (1/75)
Test group 02 (150 mg/kg bw): post-mortem autolysis (1/94), general finding - not assessed (1/94)
Test group 03 (600 mg/kg bw): general finding - not assessed (3/75)
- these findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences; therefore, these findings were not considered to be associated to the test substance

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity F1

Generation:

F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weights of the offspring during lactation

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Number of liveborn pups / number of stillborn pups:
Test group 10 (00 mg/kg bw): 10.3 / 3.7 (1 animal had all pups stillborn, the respective values reflect the normal range of biological variation inherent in the strain used)
Test group 11 (40 mg/kg bw): 10.9 / 0.8
Test group 12 (150 mg/kg bw): 10.7 / 2.0
Test group 13 (600 mg/kg bw): 11.8 / 3.4
Viability index (PND 0-4):
Test group 10 (00 mg/kg bw): 99.6%
Test group 11 (40 mg/kg bw): 100.0%
Test group 12 (150 mg/kg bw): 99.6%
Test group 13 (600 mg/kg bw): 99.3%
Lactation index (PND 4-21):
Test group 10 (00 mg/kg bw): 100.0%
Test group 11 (40 mg/kg bw): 100.0%
Test group 12 (150 mg/kg bw): 100.0%
Test group 13 (600 mg/kg bw): 100.0%

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male:
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): reduced on PND 14-21 (max. -9.8%), reduced in the total interval PND 1-21
Female:
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): reduced on PND 14-21 (max -9.5%), reduced in the total interval PND 1-21

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio
% live male pups PND 0 vs. PND 21 / % live female pups PND 0 vs. PND 21:
Test group 00 (00 mg/kg bw): 50.5 vs. 50.8 / 49.5 vs. 49.2
Test group 01 (40 mg/kg bw): 48.4 vs. 47.5 / 51.6 vs. 52.5
Test group 02 (150 mg/kg bw): 52.1 vs. 51.3 / 47.9 vs. 48.7
Test group 03 (600 mg/kg bw): 47.8 vs. 48.8 / 52.2 vs. 51.2
- no substantial difference; slight differences were regarded to be spontaneous in nature

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Males:
Test group 10 (00 mg/kg bw): 3.13 mm / 1.63 (Index Cubic Root)
Test group 11 (40 mg/kg bw): 3.13 mm / 1.64 (Index Cubic Root)
Test group 12 (150 mg/kg bw): 3.08 mm / 1.61 (Index Cubic Root)
Test group 13 (600 mg/kg bw): 3.03 mm / 1.60 (Index Cubic Root)
Females:
Test group 10 (00 mg/kg bw): 1.36 mm / 0.72 (Index Cubic Root)
Test group 11 (40 mg/kg bw): 1.36 mm / 0.72 (Index Cubic Root)
Test group 12 (150 mg/kg bw): 1.37 mm / 0.73 (Index Cubic Root)
Test group 13 (600 mg/kg bw): 1.34 mm / 0.71 (Index Cubic Root)

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Males:
Nipple development (% / number) on day 13
Test group 11 (40 mg/kg bw): 42.88% / 1.15
Test group 12 (150 mg/kg bw): 50.66% / 1.42
Test group 13 (600 mg/kg bw): 64.38% / 2.22
Nipple development (% / number) on day 20
Test group 11 (40 mg/kg bw): 0.0 / 0.0
Test group 12 (150 mg/kg bw): 0.0 / 0.0
Test group 13 (600 mg/kg bw): 0.0 / 0.0

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Spleen (absolute): 0.2457 / 0.2350 / 0.2470 / 0.2072* versus in test group 10, 11 / 12 / 13, respectively
Thymus (absolute): 0.2411 / 0.2274 / 0.2540 / 0.2195* versus in test group 10, 11 / 12 / 13, respectively
Brain (relative): 102% / 102% / 108%** versus control in test group 11 / 12 / 13, respectively
Females:
Spleen (absolute): 0.2451 / 0.2366 / 0.2380 / 0.2053** versus in test group 10, 11 / 12 / 13, respectively
Brain (relative): 100% / 100% / 107%* versus control in test group 11 / 12 / 13, respectively
Males and females combined:
Spleen (absolute): 0.2454 / 0.2368 / 0.2425 / 0.2062** versus in test group 10, 11 / 12 / 13, respectively
Spleen (relative): 100% / 100% / 92%* versus control in test group 11 / 12 / 13, respectively
Brain (relative): 101% / 101% / 107%** versus control in test group 11 / 12 / 13, respectively
* or ** = p-level of significance ≤ 0.05 or ≤ 0.01, respectively
All weight changes in the high-dose group are considered to be a consequence of the reduced body weight of the corresponding pups at weaning, and may reflect a slight delay in the development of the immune system. This is further strengthened by the fact, that there was no evidence for immunotoxicity based on the examination of lymphocyte subpopulations in the spleen and by histopathology of the spleen and thymus in cohort F0 and F1 A animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Test group 11 (40 mg/kg bw): post-mortem autolysis (1/121)
Test group 12 (150 mg/kg bw): post-mortem autolysis (2/131), dilated renal pelvis (1/131)
Test group 13 (600 mg/kg bw): post-mortem autolysis (7/141), small liver lobe (1/141)
Females:
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): post-mortem autolysis (2/121), general finding - not assessed (1/121), empty stomach (1/121)
Test group 13 (600 mg/kg bw): post-mortem autolysis (3/151), general finding - not assessed (2/151), dilated renal pelvis (1/151)
- these findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences; therefore, these findings were not considered to be associated to the test substance

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity F2

Generation:

F2

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weights of the offspring during lactation

Target system / organ toxicity (F2)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study the NOAEL (no observed adverse effect level) for general and systemic toxicity is 40 mg/kg bw/d for the F0 and 150 mg/kg bw/d for the F1 parental rats, based on reduced body weights in males at 600 mg/kg bw/d as well as clinical pathology and histopathological findings observed at 150 mg/kg bw/d (F0) and 600 mg/kg bw/d (F1).
The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 600 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity in the F1 and F2 progeny is 150 mg/kg bw/d, based on reduced body weights of the offspring during lactation at 600 mg/kg bw/d.